The overall goal of this proposal is to define the molecular mechanisms that establish the first longitudinal axon pathways in the embryonic vertebrate brain. Longitudinal axons form the main communication highways in the CNS, transmitting all signals between brain regions and spinal cord. During early brain development, pioneer axons establish an initial simple array of longitudinal tracts by choosing specific pathways and accurately growing long distances. Our preliminary results have identified Slit/Robo signaling as a major determinant of longitudinal guidance, specifically the Slit1 and Slit2 secreted signaling proteins and their Robo1 and Robo2 receptors. We show that all pioneer longitudinal tracts are severely disrupted in mouse embryos carrying Slit or Robo mutations, due to extensive dorsal-ventral wandering and other errors. The overall goal of the proposal is to define how Slit/Robo signaling controls longitudinal guidance. The experimental system is the simple organized array of pioneer axons in early mouse and chick embryos, using a range of functional assays in intact embryos and with cultured axons. Aim 1. Determine the function of secreted Slit cues in guiding longitudinal axons. Our initial analysis of Slit mutant embryos identifies Slit1 and 2 as providing critical cues. We hypothesize that Slits function either as direct long-range instructive signals to orient and position axons, or as obligatory permissive signals for axons to respond to other guidance cues. To distinguish between these mechanisms, we will study axon projections in Slit mutant mice, challenge axons with Slits in explant cultures, and mis-express Slits in vivo. Aim 2. Test the function of the Robo family of Slit receptors in longitudinal guidance. Our evidence shows that axons mutant for Robo1 and 2 are unable to navigate along precise pathways. These results indicate that Robo1 and 2 mediate Slit signaling. We propose that Robos set the position of different longitudinal tracts via modulation of either specific combinations of isoforms or by different expression levels. To test these Robo mechanisms, we will study axons in Robo mutant mice, and mis-express Robos in specific axon populations. The main goal of this proposal is to define how neural wiring can form during embryonic brain development. New insights into neuron growth mechanisms during development will provide significant insights into birth defects in the brain, as well as how functional regeneration of the nervous system could be stimulated and guided following trauma or disease.